Method of preparing carboxylic acid functionalized polymers

ABSTRACT

Methods for preparing water soluble, non-peptidic polymers carrying carboxyl functional groups, particularly carboxylic acid functionalized poly(ethylene glycol) (PEG) polymers are disclosed, as are the products of these methods. In general, an ester reagent R(C═O)OR′, wherein R′ is a tertiary group and R comprises a functional group X, is reacted with a water soluble, non-peptidic polymer POLY-Y, where Y is a functional group which reacts with X to form a covalent bond, to form a tertiary ester of the polymer, which is then treated with a strong base in aqueous solution, to form a carboxylate salt of the polymer. Typically, this carboxylate salt is then treated with an inorganic acid in aqueous solution, to convert the carboxylate salt to a carboxylic acid, thereby forming a carboxylic acid functionalized polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/922,020, filed Jun. 19, 2013, now U.S. Pat. No.10,011,682, which is a continuation application of U.S. patentapplication Ser. No. 13/279,134, filed Oct. 21, 2011, which is acontinuation application of U.S. patent application Ser. No. 13/023,960,filed Feb. 9, 2011, now U.S. Pat. No. 8,067,505, which is a continuationapplication of U.S. patent application Ser. No. 10/982,303, filed Nov.4, 2004, which claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Patent Application No. 60/517,794, filed Nov. 6,2003, all of which are incorporated herein by reference in theirentireties.

FIELD

This invention relates to methods for preparing water soluble,non-peptidic polymers carrying carboxyl functional groups, particularlycarboxylic acid functionalized poly(ethylene glycol) (PEG)polymers.

BACKGROUND

Poly(ethylene glycol) (PEG) derivatives activated with electrophilicgroups are useful for coupling to nucleophilic groups, such as aminogroups, of biologically active molecules. In particular, active estersand other carboxylic acid derivatives of PEG have been used to attachPEG to proteins bearing amino groups.

PEG molecules having terminal carboxymethyl groups have been described,for example, by Martinez et al., U.S. Pat. No. 5,681,567, Veronese etal., Journal of Controlled Release 10:145-154 (1989), and Buckmann etal., Makromol. Chem. 182(5): 1379-1384 (1981). U.S. Pat. No. 5,672,662(Harris et al.) discloses PEG derivatives having a terminal propionic orbutanoic acid moiety. Such carboxyl-terminated PEGs are used to prepareactive esters suitable for conjugation to proteins or other moleculesbearing amino groups.

However, a persistent problem associated with preparation ofcarboxyl-functionalized polymers has been the difficulty in obtainingthe desired polymer product at a sufficiently high purity level. Forexample, Veronese et al. and Buckmann et al., cited above, employ amethod of synthesizing mPEG carboxylic acids which comprises convertingmPEG-OH to an ethyl ester of mPEG carboxylic acid, by base-catalyzedreaction of mPEG-OH with an α-halo ethyl ester, followed bybase-promoted hydrolysis of the ester. However, this approach providesmPEG acids of only about 85% purity, with the main contaminant beingmPEG-OH, which cannot be separated from the mPEG carboxylic acid usingtypical purification methods such as precipitation, crystallization orextraction. Removal of mPEG-OH requires the use of preparative ionexchange column chromatography, which is time consuming and expensive.PEG carboxylic acids obtained commercially frequently contain residualamounts of PEG-OH, which complicates the preparation of derivatives orbioconjugates based on these materials.

U.S. Pat. Nos. 5,278,303, 5,605,976 and 5,681,567 report the preparationof PEG carboxylic acids containing little or no starting material (PEGalcohol) by employing a tertiary alkyl haloacetate to prepare a tertiaryalkyl ester-functionalized PEG, which is then hydrolyzed with acid,preferably trifluoroacetic acid (TFA).

Various treatises on the use of protecting groups note that tertiaryalkyl esters, such as t-butyl esters, are stable to mild base hydrolysistypically used to hydrolyze primary alkyl esters, such as ethyl esters.Strong base hydrolysis could cause cleavage of carboxylic acid groups.See, for example, T. W. Greene, Protective Groups in Organic Synthesis,3^(rd) edition, 1999, p. 406; or P. J. Kocienski, Protecting Groups,1994, p. 125. Accordingly, these tertiary alkyl esters areconventionally cleaved with acid, typically with TFA.

However, use of trifluoroacetic acid can result in purification andproduct stability problems. Trifluoroacetic acid is difficult tocompletely remove from the final carboxyl-functionalized polymer,particularly the amount of TFA suggested in the above-referencedpatents. The presence of residual trifluoroacetic acid results in poorproduct stability, due to degradation of the polymer caused byacid-promoted autoxidation. See, for example, M. Donbrow, “Stability ofthe Polyoxyethylene Chain”, in Nonionic Surfactants: Physical Chemistry,M. J. Schick, ed., Marcel Dekker, 1987, pp. 1011 ff. This articlereports that acids catalyze the formation of hydroperoxides andhydroperoxide rupture, leading to cleavage of polyoxyethylene chains.

Although U.S. Pat. No. 5,605,976 suggests distillation as a means forseparating organic materials from the polymer product, even compoundswith very low boiling points are difficult to remove from high molecularweight polymers using a distillation process, and the difficultyincreases as the molecular weight of the polymer increases.

There is a need in the art for alternative methods for preparingcarboxylic acid functionalized polymers in high yield and free fromsignificant amounts of polymer contaminants, particularly the polymerstarting material. There is also a need in the art for alternativesynthesis methods that do not utilize reagents that are either difficultto remove from the final polymer product or cause product stabilityproblems.

SUMMARY

In one aspect, the invention provides a method for preparing a watersoluble, non-peptidic polymer functionalized with a carboxyl group, themethod comprising:

(i) reacting an ester reagent R(C═O)OR′, where R′ is a tertiary groupand R comprises a functional group X, with a water soluble, non-peptidicpolymer POLY-Y, where Y is a functional group which reacts with X toform a covalent bond, to form a tertiary ester of the polymer; and

(ii) treating the tertiary ester of the polymer with a strong base, suchas an alkali metal hydroxide, in aqueous solution, to form a carboxylatesalt of the polymer. The method may further comprise the step of (iii)treating the carboxylate salt of the polymer with an inorganic acid inaqueous solution, to convert the carboxylate salt to a carboxylic acid,thereby forming a carboxylic acid functionalized polymer. The carboxylicacid functionalized polymer can then be extracted from the aqueoussolution with a suitable solvent, preferably a chlorinated solvent.

In one embodiment, X is a leaving group, such as a halide or a sulfonateester, and Y is a hydroxyl group. When Y is a hydroxyl group, thereaction (i) is preferably carried out in the presence of a base, e.g. abase of the form R′O⁻M⁺, where M+ is a cation.

The treatment with strong base in reaction (ii) is preferably effectiveto produce a reaction pH of about 11 to 13. The inorganic acid, e.g. amineral acid, in step (iii) is preferably an acid that producesnon-nucleophilic anions in aqueous solution. Preferred acids includesulfuric acid, nitric acid, phosphoric acid, and hydrochloric acid. Theacid treatment of (iii) is preferably effective to produce a reaction pHof about 1 to 3.

The tertiary ester reagent employed in reaction (i) preferably has thestructure (I):

In structure (I), X is a leaving group; and each of R¹ and R² isindependently selected from hydrogen, alkyl, cycloalkyl, alkoxy, aryl,aralkyl, and heterocycle. Preferably, the group (CR¹R²)_(n) does notinclude two heteroatoms attached to the same carbon atom; for example,R¹ and R² on the same carbon atom are preferably not both alkoxy. Eachof R³-R⁵ is independently selected from lower alkyl, aryl, aralkyl, andcycloalkyl, where any of R³-R⁵ may be linked to form a ring or ringsystem, such as adamantyl. Any of R¹ to R⁵, excepting hydrogen, may besubstituted with a group selected from lower alkyl, lower alkoxy, C3-C6cycloalkyl, halo, cyano, oxo(keto), nitro, and phenyl. The variable n is1 to about 24, preferably 1 to 6, more preferably 1 to 4, and mostpreferably 1 or 2. In one embodiment, n is 1.

In selected embodiments of structure (I), each of R¹ and R² isindependently hydrogen or unsubstituted lower alkyl, preferably hydrogenor methyl, and each of R³ to R⁵ is independently unsubstituted loweralkyl or phenyl, preferably methyl, ethyl, or phenyl. In one embodiment,each of R¹ and R² is H and n is 1.

The leaving group X in structure (I) is preferably a halide or asulfonate ester. In one embodiment, the tertiary ester reagent is atertiary alkyl haloacetate, such as a t-butyl haloacetate.

The water soluble, non-peptidic polymer is preferably selected from thegroup consisting of poly(alkylene glycols), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazolines, poly(N-acryloylmorpholine), and copolymers orterpolymers thereof. In a preferred embodiment, the polymer is apoly(ethylene glycol). The poly(ethylene glycol) may be linear andterminated at one end with the functional group Y and at the other endwith another functional group Y′ or a capping group, such as a methoxygroup. Alternatively, the poly(ethylene glycol) may be branched, forked,or multiarmed.

The method may further comprise converting the carboxylic acid of thecarboxylic acid functionalized polymer to an activated carboxylic acidderivative, e.g. an activated ester, such as, for example, anN-succinimidyl ester, o-, m-, or p-nitrophenyl ester, 1-benzotriazolylester, imidazolyl ester, or N-sulfosuccinimidyl ester. The polymer canthen be conjugated with a biologically active molecule, by reacting thecarboxylic acid derivative with a functional group, preferably anucleophilic group such as a hydroxyl, thiol, or amino group, on thebiologically active molecule. Preferably, the nucleophilic group is anamino group.

In a preferred embodiment of the method, as noted above, the polymer isa PEG polymer. In this aspect, the invention provides a method forpreparing a poly(ethylene glycol) (PEG) functionalized with a carboxylgroup, the method comprising:

(i) reacting a tertiary ester reagent R(C═O)OR′, where R′ is a tertiaryalkyl group and R comprises a functional group X, with a polymer PEG-Y,where Y is a functional group which reacts with X to form a covalentbond, to form a PEG tertiary ester; and

(ii) treating the PEG tertiary ester with a strong base, such as analkali metal hydroxide, in aqueous solution, to form a PEG carboxylatesalt. The method may further comprise (iii) treating the PEG carboxylatesalt with an inorganic acid in aqueous solution, to convert thecarboxylate salt to a carboxylic acid, thereby forming a PEG carboxylicacid. Preferred embodiments of the method correspond to those describedabove. The method may further comprise converting the PEG-carboxylicacid to an activated carboxylic acid derivative, such as an activatedester, and conjugating the polymer to a biologically active molecule, byreacting the carboxylic acid derivative with a functional group on themolecule, as described above.

In one embodiment, the poly(ethylene glycol) is linear and is terminatedat one end with the functional group Y and at the other end with anotherfunctional group Y′ or with a capping group, such as a methoxy group.The molecular weight of the PEG is preferably in the range of about 100Da to about 100 kDa, more preferably in the range of about 300 Da toabout 40, 50, or 60 kDa. In other embodiments, the PEG is branched,forked, or multiarmed, as described further below.

In a related aspect, the invention provides an isolated polymer productcomprising a carboxylic acid functionalized polymer, made by the methoddisclosed herein, wherein the product contains less than 5% by weight ofthe starting material; that is, the POLY-Y or PEG-Y polymer, with thebalance consisting essentially of the carboxylic acid functionalizedpolymer. Preferably, the isolated polymer product contains less than 2%,more preferably less than 1%, and most preferably less than 0.5% byweight of POLY-Y or PEG-Y polymer. In further preferred embodiments, theisolated polymer product contains less than 0.4%, more preferably lessthan 0.3%, and most preferably less than 0.2% by weight of POLY-Y orPEG-Y polymer.

In a further preferred aspect, the isolated polymer product containssubstantially no amount of low molecular weight organic acid. In oneembodiment, the isolated polymer product contains substantially noamount of monomeric organic carboxylic acid, such as trifluoroaceticacid.

In one embodiment of the polymer product of the invention, thecarboxylic acid functionalized polymer is a PEG carboxylic acid. Forexample, the carboxylic acid functionalized polymer may bemPEG-CH₂—COOH, and contains less than 5%, preferably less than 2%, morepreferably less than 0.5%, and most preferably less than 0.2% by weightof mPEG-OH. Preferably, the product contains substantially no amount oftrifluoroacetic acid.

In another embodiment of the product, the carboxylic acid functionalizedpolymer is HOOC—CH₂-PEG-CH₂—COOH, and contains less than 5%, preferablyless than 2%, more preferably less than 0.5%, and most preferably lessthan 0.2% by weight of HO-PEG-OH. Preferably, the product containssubstantially no amount of trifluoroacetic acid.

In a further embodiment of the product, the carboxylic acidfunctionalized polymer is a multifunctional branched or multiarmcarboxylic acid functionalized PEG represented by PEG-(CH₂—COOH)_(x),where x is 3 to 8, and contains less than 5%, preferably less than 2%,more preferably less than 0.5%, and most preferably less than 0.2% byweight of PEG-(OH)_(x). Preferably, the product contains substantiallyno amount of trifluoroacetic acid.

The invention further provides an improvement in a method of preparing apoly(ethylene glycol) (PEG)polymer functionalized with a carboxyl group,by reaction of a tertiary ester reagent R(C═O)OR′, where R′ is atertiary alkyl group and R comprises a functional group X, with apolymer PEG-Y, where Y is a functional group which reacts with X to forma covalent bond, to form a PEG tertiary ester. The improvement comprisestreating the PEG tertiary ester with a strong base, preferably an alkalimetal hydroxide, in aqueous solution, to form a PEG carboxylate salt.The strong base is preferably one that is strong base is effective toproduce a reaction pH of about 11 to 13 in the aqueous solution.

The improved method may further comprise treating the PEG carboxylatesalt with an inorganic acid in aqueous solution, to convert thecarboxylate salt to a carboxylic acid, thereby forming a PEG carboxylicacid. The inorganic acid is preferably a mineral acid selected from thegroup consisting of sulfuric acid, nitric acid, phosphoric acid, andhydrochloric acid.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the included drawings.

DETAILED DESCRIPTION

The present invention now will be described more fully. This inventionmay, however, be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art. The invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like set forth inthis description, as such may vary within the scope of the invention asembodied by the appended claims. The terminology used herein is fordescribing particular embodiments only, and is not intended to belimiting.

I. Definitions

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

As used herein, “non-peptidic” refers to a polymer substantially free ofpeptide linkages. However, the polymer may include a minor number ofpeptide linkages spaced along the length of the backbone, such as, forexample, no more than about 1 peptide linkage per about 50 monomerunits.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:—O(CH₂CH₂O)_(m)— or —CH₂CH₂O(CH₂CH₂O)_(m)— CH₂CH₂—, where m is generallyfrom 3 to about 3000. In a broader sense, “PEG” can refer to a polymerthat contains a majority, i.e. greater than 50%, of subunits that are—CH₂CH₂O—.

The terminal groups and architecture of the overall PEG may vary. ThePEG may contain an end-capping group on a terminal oxygen which isgenerally a carbon-containing group typically comprised of 1-20 carbonsand is preferably selected from alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, heterocyclo, and substituted forms of any of the foregoing.The end-capping group can also be a silane. Most preferred arealkyl(alkoxy) or aralkyl(aralkoxy) capping groups, such as methyl, ethylor benzyl.

The end-capping group can also advantageously comprise a detectablelabel. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

The other (“non-end-capped”) terminus is a typically hydroxyl, amine oran activated group that can be subjected to further chemicalmodification.

Specific PEG forms for use in the invention include PEGs having avariety of molecular weights, structures or geometries (e.g., branched,linear, forked, multiarmed).

A “multifunctional” polymer has 3 or more functional groups, which maybe the same or different. Multifunctional polymers will typicallycontain from about 3-100 functional groups, or from 3-50 functionalgroups, or from 3-25 functional groups, or from 3-15 functional groups,or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9or 10 functional groups.

A “difunctional” polymer has two functional groups contained therein,which may be the same (i.e., homodifunctional) or different (i.e.,heterodifunctional).

“Molecular mass” or “molecular weight” refers to the average molecularmass of a polymer, typically determined by size exclusionchromatography, light scattering techniques, or intrinsic velocitydetermination in 1,2,4-trichlorobenzene. Unless otherwise noted,molecular weight is expressed herein as number average molecular weight(M_(n)), which is defined as ΣNiMi/ΣNi, wherein Ni is the number ofpolymer molecules (or the number of moles of those molecules) havingmolecular weight Mi.

The polymers of the invention, or employed in the invention, aretypically polydisperse; i.e., the number average molecular weight andweight average molecular weight of the polymers are not equal. Thepolydispersity values, expressed as a ratio of weight average molecularweight (Mw) to number average molecular weight (Mn), (Mw/Mn), aregenerally low; that is, less than about 1.2, preferably less than about1.15, more preferably less than about 1.10, still more preferably lessthan about 1.05, yet still most preferably less than about 1.03, andmost preferably less than about 1.025.

An “activated carboxylic acid” refers to a functional derivative of acarboxylic acid that is more reactive than the parent carboxylic acid,particularly with respect to nucleophilic attack. Activated carboxylicacids include but are not limited to acid halides (such as acidchlorides), anhydrides, and esters.

More generally, the term “activated” or “reactive”, when used inconjunction with a particular functional group, refers to a functionalgroup that reacts readily with an electrophile or a nucleophile onanother molecule, in contrast to groups that require strong catalysts orimpractical reaction conditions in order to react (i.e., “nonreactive”or “inert” groups).

The term “protecting group” or “protective group” refers to a moietythat prevents or blocks reaction of a particular chemically reactivefunctional group in a molecule under certain reaction conditions. Theprotecting group will vary depending upon the type of chemicallyreactive group being protected as well as the reaction conditions to beemployed and the presence of additional reactive or protecting groups inthe molecule, if any. Protecting groups known in the art can be found inGreene, T. W., et al., Protective Groups in Organic Synthesis, 3rd ed.,John Wiley & Sons, New York, N.Y. (1999). As used herein, the term“functional group” or any synonym thereof is meant to encompassprotected forms thereof.

The term “spacer” or “spacer moiety” refers to an atom or a collectionof atoms used to link interconnecting moieties, such as a terminus of awater-soluble polymer portion and an electrophile. A typical spacerincludes bonds selected from alkylene (carbon-carbon), ether, amino,amide, ester, carbamate, urea, and keto, and combinations thereof. Aspacer may include short alkylene moieties alternating with, or flankedby, one or more types of heteroatom-containing linkages listed above.Various examples include —CH₂OCH₂CH₂CH₂—, —CH₂C(O)NHCH₂—, —C(O)OCH₂—,—OC(O)NHCH₂CH₂—, —CH₂CH₂NHCH₂, —CH₂CH₂C(O)CH₂CH₂—,—CH₂CH₂CH₂C(O)NHCH₂CH₂NH—, and —CH₂CH₂CH₂C(O)NHCH₂CH₂NHC(O)CH₂CH₂—. Thespacer moieties of the invention may be hydrolytically stable or mayinclude a physiologically hydrolyzable or enzymatically degradablelinkage (e.g. an ester linkage).

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or, preferably, linear(unbranched). Exemplary alkyl groups include ethyl, propyl, butyl,pentyl, 2-methylbutyl, 2-methylpropyl(isobutyl), 3-methylpentyl, and thelike. As used herein, “alkyl” includes cycloalkyl when three or morecarbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup having 2 to 15 carbon atoms and containing at least one doublebond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group having 2 to 15 atoms and containing at least onetriple bond, such as ethynyl, n-propynyl, isopentynyl, n-butynyl,octynyl, decynyl, and so forth.

“Alkoxy” refers to an —OR group, wherein R is alkyl or substitutedalkyl, preferably C1-C20 alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),more preferably lower alkyl (i.e. C1-C6).

“Aryl” refers to a substituted or unsubstituted monovalent aromaticradical having a single ring (e.g., phenyl) or two condensed or fusedrings (e.g., naphthyl). Multiple aryl rings may also be unfused (e.g.biphenyl). The term includes heteroaryl groups, which are aromatic ringgroups having one or more nitrogen, oxygen, or sulfur atoms in the ring,such as furyl, pyrrole, pyridyl, and indole.

“Aralkyl” refers to an alkyl, preferably lower (C₁-C₄, more preferablyC₁-C₂) alkyl, substituent which is further substituted with an arylgroup; examples are benzyl and phenethyl. “Aralkoxy” refers to a groupof the form —OR where R is aralkyl; one example is benzyloxy.

A “heterocycle” refers to a ring, preferably a 5- to 7-membered ring,whose ring atoms are selected from the group consisting of carbon,nitrogen, oxygen and sulfur. Preferably, the ring atoms include 3 to 6carbon atoms. Examples of aromatic heterocycles (heteroaryl) are givenabove; non-aromatic heterocycles include, for example, pyrrolidine,piperidine, piperazine, and morpholine.

A “substituted” group or moiety is one in which a hydrogen atom has beenreplaced with a non-hydrogen atom or group, which is preferably anon-interfering substituent.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule. These include, but are not limited to,lower alkyl, alkenyl, or alkynyl; lower alkoxy; C3-C6 cycloalkyl; halo,e.g., fluoro, chloro, bromo, or iodo; cyano; oxo (keto); nitro; andphenyl.

A “tertiary group” is a group of the form —CR₃, where each R is anorganic moiety linked to C via a carbon atom. Each R may be, forexample, alkyl, cycloalkyl, aryl, or aralkyl, substituted orunsubstituted. Examples of tertiary groups include t-butyl, where each Ris methyl; triphenylmethyl (trityl), where each R is phenyl; anddimethoxytrityl (DMT), where two R's are p-methoxyphenyl and one isphenyl. Also included are groups where one or more R's form a ring orring system, such as adamantyl.

A “tertiary ester” is an ester having a tertiary group as its alcoholportion; i.e. R′—(C═O)—OCRs, where CR₃ is a “tertiary group” as definedabove, and R′ is the acid portion of the ester.

A “carboxyl group” as used herein refers to the group —C(═O)OH(carboxylic acid) or —C(═O)O⁻M⁺, where M⁺ is a positively charged ion,such as an alkali metal ion (carboxylate group).

A “low molecular weight” organic acid refers to an acidic organiccompound having a molecular weight less than about 400, preferably lessthan about 300, and more preferably less than about 200. The termtypically refers to a non-polymeric and non-oligomeric acid, andgenerally refers to an acid used as a reagent. Examples include formicacid, acetic acid, trifluoroacetic acid (TFA), and p-toluenesulfonicacid.

An “electrophile” is an atom or collection of atoms having anelectrophilic center, i.e., a center that is electron-seeking or capableof reacting with a nucleophile.

A “nucleophile” refers to an atom or a collection of atoms having anucleophilic center, i.e., a center that is seeking an electrophiliccenter or capable of reacting with an electrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include, butare not limited to, carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, and orthoesters.

An “enzymatically degradable linkage” is a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water; i.e.it does not undergo hydrolysis under physiological conditions to anyappreciable extent over an extended period of time. Generally, ahydrolytically stable linkage is one that exhibits a rate of hydrolysisof less than about 1-2% per day under physiological conditions. Examplesof hydrolytically stable linkages include carbon-carbon bonds, ethers,amines, and amides. Hydrolysis rates of representative chemical bondscan be found in most standard chemistry textbooks.

A product containing “substantially no amount” of a specified componenteither contains no amount of the specified component, or contains anamount which is undetectable by conventional methods of analysis of theproduct, and/or has no detectable effect on the properties or stabilityof the product. For example, a product which has never knowingly ordeliberately been exposed to or contacted with a particular substancewould be considered to contain substantially no amount of the substance.

Each of the terms “drug,” “biologically active molecule,” “biologicallyactive moiety,” and “biologically active agent”, when used herein, meansany substance which can affect any physical or biochemical property of abiological organism, where the organism may be selected from viruses,bacteria, fungi, plants, animals, and humans. In particular, as usedherein, biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like. Also included are foods,food supplements, nutrients, nutriceuticals, drugs, vaccines,antibodies, vitamins, and other beneficial agents.

The term “conjugate” refers to an entity formed as a result of covalentattachment of a molecule, e.g., a biologically active molecule, to areactive polymer molecule, preferably a poly(ethylene glycol).

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to a patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and therapeutically effective amount” are used herein to referto mean the amount of a polymer-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

The term “patient” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of abiologically active agent or conjugate thereof, and includes both humansand animals.

II. Method of Preparing Carboxylic Acid Functionalized Polymers

A. Overview

The present invention provides, in one aspect, a method of preparing awater soluble, non-peptidic polymer functionalized with a carboxylgroup, i.e. a carboxylate salt or carboxylic acid. The method involvesreacting a tertiary ester reagent R(C═O)OR′, where R′ is a tertiarygroup, as defined above, and R includes a functional group X, with awater soluble, non-peptidic polymer POLY-Y, where Y is a functionalgroup which reacts with X to form a covalent bond, to form a tertiaryester of the polymer, which may be represented as POLY-R—(C═O)OR′. Thenature of the linkage between POLY and R depends on the functionalgroups Y and X.

The starting material of the reaction, represented by POLY-Y, or byPEG-Y when the polymer is a polyethylene glycol, may include more thanone functional group Y, in various configurations. Examples includelinear, branched, and multiarmed PEGs containing multiple hydroxylgroups, as discussed further below. The product of the reaction, i.e.the carboxyl-functionalized polymer, contains a number of carboxylgroups which is equal to the number of functional groups Y in thestarting material (or greater than Y, if the starting material hasexisting carboxyl groups).

Preferably, the functional group Y of the polymer is a hydroxyl group,or other nucleophilic group, and the functional group X of the tertiaryester reagent is a leaving group capable of being displaced by Y. Otherpossible functional group combinations are described below.

Once the tertiary ester group is attached to the polymer, it isconverted to a carboxylate by base hydrolysis in aqueous solution, whichis preferably followed by acidification to produce the carboxylic acid.Surprisingly, it has been found that the tertiary ester, while stable inthe presence of the base used in the initial nucleophilic substitutionreaction, can be removed by base-promoted hydrolysis. As noted above,tertiary alkyl esters, such as t-butyl esters, are conventionallythought to be resistant to base hydrolysis.

The general reaction scheme below depicts a preferred embodiment of themethod of the invention, where Y is hydroxyl and X is a leaving group,and the ester reagent has the structure shown as (I).

B. Reaction Components

In the preferred ester reagent (I), each of R¹ and R² is independentlyselected from H, lower alkyl, cycloalkyl, alkoxy, aryl, aralkyl, andheterocycle; and each of R³-R⁵ is independently selected from loweralkyl, aryl, and aralkyl, each as defined above. Preferably, the group(CR¹R²)_(n) does not include two heteroatoms attached to the same carbonatom; for example, R¹ and R² on the same carbon atom are preferably notboth alkoxy. Any of R¹ to R⁵, excepting hydrogen, may be substitutedwith a non-interfering substituent, as defined above.

Preferably, each of R¹ and R² is independently hydrogen or unsubstitutedlower alkyl, and each of R³ to R⁵ is independently unsubstituted loweralkyl or phenyl. In selected embodiments, each of R¹ and R² isindependently hydrogen or methyl, more preferably hydrogen, and each ofR³ to R⁵ is independently methyl, ethyl, or phenyl.

The variable n is 1 to about 24, preferably 1 to about 12. In selectedembodiments, n is 1 or 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to8, 1 to 9, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, or 1 to 24. Infurther selected embodiments, n is 1 to 6; preferably, n is 1 to 4; andmore preferably, n is 1 or 2. When n is greater than 1, the moiety—(CR¹R²)n-preferably includes at most two, and more preferably at mostone, non-hydrogen embodiment of R¹ or R².

In further embodiments, n is 1, and R¹ and R² are independently hydrogenor methyl. In one such embodiment, when both R¹ and R² are hydrogen, theproduct (IV) contains a carboxymethyl group.

Preferably, the functional group X on the ester reagent (II) is aleaving group, such as halo, e.g. chloro or bromo, or sulfonate ester,such as p-toluenesulfonyl (tosyl), methanesulfonyl (mesyl),trifluorosulfonyl, or trifluoroethylsulfonyl (tresyl). However, otherfunctional groups capable of reacting with a functional group on thepolymer, to form a covalent linkage, could also be used. Preferably, thefunctional group on the polymer is a nucleophilic group, such as amine,hydrazide (—C(═O)NHNH₂), or thiol, and the functional group X on theester reagent is an electrophilic group. In addition to leaving groupssuch as those described above, electrophilic groups include carboxylicester, including imide ester, orthoester, carbonate, isocyanate,isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,thiosulfonate, silane, alkoxysilane, halosilane, and phosphoramidate.More specific examples of these groups include succinimidyl ester orcarbonate, imidazolyl ester or carbonate, benzotriazole ester orcarbonate, p-nitrophenyl carbonate, vinyl sulfone, chloroethylsulfone,vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, and dione.Also included are other activated carboxylic acid derivatives, as wellas hydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal). Preferred electrophilic groups includesuccinimidyl carbonate, succinimidyl ester, maleimide, benzotriazolecarbonate, glycidyl ether, imidazoyl ester, p-nitrophenyl carbonate,acrylate, aldehyde, and orthopyridyl disulfide.

In general, the functional group X on the reagent is chosen such that itreacts with the functional group Y on the polymer much more readily thanthe functional group Y would react with the t-butyl ester portion of thereagent. When the polymeric functional group Y is a nucleophile, such ashydroxyl, X is most suitably a good leaving group such as halo orsulfonate ester.

Particularly preferred ester reagents include t-butyl haloacetates, suchas t-butyl bromoacetate, t-butyl chloroacetate, and t-butyl iodoacetate.Such t-butyl haloacetates are available, for example, from SigmaChemical Co., St. Louis, Mo.

In Scheme I, POLY-OH is a water soluble, non-peptidic polymer, such as,for example, mPEG-OH. In general, the polymer can be any water soluble,non-peptidic polymer, having any available geometric configuration(e.g., linear, branched, forked, etc.), as discussed further below. Forthe sake of simplicity, the reaction scheme given above utilizes apolymer with a single hydroxyl group. However, as would be appreciatedby one of ordinary skill in the art, the polymer may comprise more thanone hydroxyl group, such as 1 to about 25 hydroxyl groups (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more hydroxyl groups). Also, the hydroxylgroup could be replaced with any nucleophilic functional group reactivewith the functional group X on the tertiary ester reagent. Suchnucleophilic functional groups include thiols, amines, and stabilizedcarbanions.

C. Reaction Process

For the first stage of the process, shown in the top line of exemplaryScheme I above, the components are preferably dissolved in a suitableorganic solvent, such as t-butanol, benzene, toluene, xylenes,tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO)and the like.

As shown in the embodiment of the invention represented by Scheme I,reaction of a polymeric hydroxyl group with the tertiary ester reagentis typically carried out in the presence of a base. Exemplary basesinclude potassium t-butoxide, butyl lithium, sodium amide, and sodiumhydride. Other strong bases could also be used.

The reaction is typically carried out at a temperature of about 0-120°C., preferably about 20-80° C., more preferably about 25-50° C.,although reaction conditions will vary based on the polymer and thefunctional groups reacting. As shown in the Examples provided below,reactions of hydroxy-containing PEGs with t-butyl bromoacetate wereeffectively carried out at temperatures between room temperature andabout 45° C.

The reaction time is typically about 0.5 hours to about 24 hours; e.g.about 1 to 20, 3 to 18, 4 to 12, or 6 to 8 hours. Typical reaction timesfor reaction of a hydroxylated polymer with t-butyl bromoacetate, asshown in the Examples below, are in the range of 12 to 20 hours. Thereaction may be monitored for completion according to standard methods.Preferably, the reaction is carried out under an inert atmosphere suchas nitrogen or argon.

The reaction preferably employs a molar excess of the ester reagent(e.g., a twofold, threefold, 6 fold, 10 fold, or 20 fold, up to about 30fold molar excess), in order to ensure that complete conversion of thepolymer starting material is achieved. Following this stage of thereaction, the organic solvent is removed, typically by evaporation ordistillation.

The ester-containing product (III) is dissolved in water, preferablydistilled or deionized water, for the second stage of the process, inwhich the ester-containing polymer is subjected to base-promotedhydrolysis by treatment with a strong base, such as hydroxide, inaqueous solution. The base hydrolysis is typically carried out at a pHto about 9 or above, preferably about 10 or above, and more preferablyabout 11 or above (e.g., about 11 to about 13). Accordingly, the base isone that is strong enough to produce a pH in this range in aqueoussolution. In one embodiment, the pH is adjusted to fall in the rangefrom about 12 to about 12.5. Preferably, base is added as necessarythroughout the reaction to maintain the pH in this range. The base isalso effective to hydrolyze any remaining ester reagent.

The base should produce a highly water soluble salt when neutralizedwith acid in the step subsequent to hydrolysis. Preferred are alkalimetal hydroxides, such as sodium hydroxide (NaOH) or potassium hydroxide(KOH).

The use of distilled or deionized water, or water having no detectablelevels of divalent cations such as calcium and magnesium ions, is alsopreferred. The base hydrolysis step is typically conducted at atemperature of about 0-50° C., preferably about 10-30° C. The reactiontime is typically about 12 to 36 hours; e.g. about 18 to 24 hours.

The polymer carboxylate salt produced by the base hydrolysis can beisolated and stored as the salt, or, preferably, it is directlyconverted to the carboxylic acid by treatment with acid, as describedbelow. Generally, the carboxylic acid is more suitable for furtherderivatization than the carboxylate salt.

The carboxylate-containing polymer is treated with aqueous acid toconvert the salt to the free acid form. The acid is preferably one thatproduces a non-nucleophilic anion in aqueous solution. Mineral acids(i.e., inorganic acids) are preferred, such as sulfuric acid, nitricacid, phosphoric acid, hydrochloric acid, and the like. Typically,sufficient acid is added to adjust the pH of the solution to about 1-3,more preferably about 2-3, which is effective to convert the polymercarboxylate salt to a free acid form, as well as to neutralize (andconvert to a freely water soluble salt) any base remaining in solution.The acidification step is typically conducted at a temperature of about0° to about 50° C., preferably about 10° to about 30° C.

The carboxylic acid-containing polymer is then separated using aconventional organic extraction step, preferably employing a halogenatedsolvent such as dichloromethane or chloroform. The polymer product isextracted into the organic phase, while any hydrolyzed reagent andexcess mineral acid or its salt remains in the aqueous phase. Thus,separation of the mineral acid from the polymer product is relativelysimple.

The organic extract is dried and concentrated, and the polymeric productis then purified using standard methods. For example, the polymer may beisolated by precipitation, followed by filtration and drying. The choiceof precipitating solvent will depend on the nature of the polymer, forPEG polymers, as described in the Examples below, ethyl ether is asuitable precipitating solvent. Recrystallization from solvents such asethyl acetate or ethanol can also be used for purification.

D. Reaction Products

Using the method of the invention, carboxyl-functionalized polymers areproduced with high purity, typically with a purity of at least about95%, preferably at least about 96%, 97%, or 98%, more preferably atleast about 99%, and most preferably at least about 99.5% by weight. Inselected embodiments, the polymer product contains at least about 99.6%,99.7%, 99.8%, or 99.9% by weight of the desired carboxyl-functionalizedpolymer. Accordingly, the product of the synthetic method disclosedherein contains less than 5%, preferably less than 4%, 3%, or 2%, morepreferably less than 1%, and most preferably less than 0.5% by weight ofstarting polymer (e.g., mPEG-OH, PEG diol, or multifunctional PEGpolyol) or other polymeric impurities. In selected embodiments, theproduct contains less than 0.4%, 0.3%, 0.2% or 0.1% by weight ofpolymeric starting material (e.g., mPEG-OH) or other polymericimpurities.

By “product” or “polymer product” is meant the material obtained bycarrying out the synthetic process disclosed above, including routineworkup procedures such as extraction, precipitation and removal ofsolvent. As shown in the Examples below, reaction mixtures containingthe products of the methods disclosed herein were worked up byextraction with a chlorinated solvent, followed by precipitation of theproduct from ethyl ether. Ion exchange chromatographic analysis of theseproducts showed essentially 100% of the desired PEG-carboxylic acidproduct, with no detectable amount of starting material or otherpolymeric impurity present. Accordingly, polymer products having theabove-disclosed purities are obtained without the need for removal ofpolymeric impurities, such as starting material. These products canoften be used directly for further derivatization and/or conjugation, asdescribed below. A further advantage of this process is that it provideshigh purity polymeric carboxylic acids, such as mPEG carboxylic acids,starting from inexpensive starting materials such as mPEG-OH, incontrast to the use of commercially available polymeric carboxylicacids, which tend to be expensive and frequently contain residualamounts of polymeric hydroxyl compound as well.

As described above, the reagents employed in the synthetic processdisclosed herein are readily removed from the polymeric product. Inparticular, no low molecular weight organic acids, such as TFA, are usedin the process. Accordingly, the carboxyl-containing polymer products ofthis invention contain no trace amounts of low molecular weight organicacids, such as TFA, as would commonly occur in polymeric carboxylicacids made using a hydrolysis process which employs such a reagent. Thepresent products therefore do not suffer the disadvantage of reducedstability associated with the presence of residual acids, as describedabove. For example, the polymer described in Example 4 below showed nosign of degradation (by GPC analysis) after 8 months of storage at −20°C.

III. Suitable Water-Soluble Non-Peptidic Polymers

Any of a variety of non-peptidic, water soluble polymers can be used inthe present invention. The polymer should be non-toxic andbiocompatible, meaning that it is capable of coexistence with livingtissues or organisms without causing harm. Examples of suitable polymersinclude, but are not limited to, poly(alkylene glycols), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazolines, poly(N-acryloylmorpholine), such as described in U.S.Pat. No. 5,629,384, which is incorporated by reference herein in itsentirety, and copolymers, terpolymers, and mixtures thereof.

The molecular weight of the polymer will vary depending on the desiredapplication, the configuration of the polymer structure, the degree ofbranching, and the like. Generally, polymers having a molecular weightof about 100 Da to about 100,000 Da are useful in the present invention,preferably about 200 Da to about 60,000 Da, and more preferably about300 Da to about 40,000 Da. Exemplary polymer embodiments have amolecular weight of approximately 200 Da, 350 Da, 550 Da, 750 Da, 1,000Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 7,500 Da, 10,000 Da, 15,000Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 50,000 Da,55,000 Da, and 60,000 Da.

The polymer preferably comprises at least one hydroxyl group, capable ofreacting with a tertiary ester reagent carrying a leaving group, asdescribed herein, in a nucleophilic substitution reaction. However,other functional groups capable of reacting with a functional group ofthe tertiary ester reagent could also be used. These include othernucleophilic groups, such as amine, hydrazide (—C(═O)NH₂), and thiol;and electrophilic groups, such as carboxylic ester, including imideester, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde,ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone,maleimide, disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane,alkoxysilane, halosilane, and phosphoramidate. More specific examples ofthese groups include succinimidyl ester or carbonate, imidazolyl esteror carbonate, benzotriazole ester or carbonate, p-nitrophenyl carbonate,vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide,iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate. Alsoincluded are other activated carboxylic acid derivatives, as well ashydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal). Preferred electrophilic groups includesuccinimidyl carbonate, succinimidyl ester, maleimide, benzotriazolecarbonate, glycidyl ether, imidazoyl ester, p-nitrophenyl carbonate,acrylate, tresylate, aldehyde, and orthopyridyl disulfide.

The functional groups are selected such that a nucleophilic group on thepolymer reacts with an electrophilic group on the tertiary esterreagent, or vice versa. The reaction between the two functional groupsis preferably a displacement reaction of a leaving group by anucleophile, but could also be, for example, a condensation or additionreaction.

The polymer preferably comprises at least one nucleophilic group, suchas a hydroxyl group. For ease of reference, hydroxyl groups arediscussed below, although other functional groups could be used. Apolymer may also include different functional groups within the samemolecule. Preferably these have similar functionality, e.g. bothnucleophilic, such as a hydroxyl group and an amino group.

Preferably, the polymer is a poly(ethylene glycol) (i.e., PEG) polymer.As noted above, the term PEG includes poly(ethylene glycol) in any of anumber of geometries or forms, including linear forms, branched ormulti-arm forms (e.g., forked PEG or PEG attached to a polyol core),pendant PEG, or PEG with degradable linkages therein.

The number and position of hydroxyl groups (and/or other functionalgroups) carried by the polymer may vary. Typically, the polymercomprises 1 to about 25 hydroxyl groups, preferably 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 hydroxyl groups. Linear polymers, such as linear PEGpolymers, typically comprise one or two hydroxyl groups, each positionedat a terminus of the polymer chain. If the PEG polymer is monofunctional(i.e., mPEG), the polymer includes a single hydroxyl group. If the PEGpolymer is difunctional, the polymer contains two hydroxyl groups, oneat each terminus of the polymer chain, or it contains a single hydroxylgroup and a different functional group at the opposing terminus.Multi-arm or branched polymers may comprise a greater number of hydroxylgroups.

Multi-armed or branched PEG molecules are described, for example, inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety. Generally speaking, a multi-armed or branched polymerpossesses two or more polymer “arms” extending from a central branchpoint, which preferably comprise a hydrolytically stable linkingstructure. An exemplary branched PEG polymer is methoxy poly(ethyleneglycol) disubstituted lysine.

In another multi-arm embodiment, the polymer comprises a central coremolecule derived from a polyol or polyamine, the central core moleculeproviding a plurality of attachments sites suitable for covalentlyattaching polymer arms to the core molecule in order to form a multi-armpolymer structure. Depending on the desired number of polymer arms, thepolyol or polyamine will typically comprise 3 to about 25 hydroxyl oramino groups, preferably 3 to about 10, most preferably 3 to about 8(e.g., 3, 4, 5, 6, 7, or 8).

Multi-armed polymers are further described, for example, in co-ownedU.S. Patent Appn. Nos. 2002/0156047 and 2002/0156047, which areincorporated herein by reference.

The PEG polymer may alternatively comprise a forked PEG. Generallyspeaking, a polymer having a forked structure is characterized as havinga polymer chain attached to two or more functional groups via covalentlinkages extending from a hydrolytically stable branch point in thepolymer. U.S. Pat. No. 6,362,254, the contents of which are incorporatedby reference herein, discloses various forked PEG structures capable ofuse in the present invention.

The PEG polymer may also be a pendant PEG molecule, having reactivegroups (e.g., hydroxyl groups) covalently attached along the length ofthe PEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

Different polymers can be incorporated into the same polymer backbone.For example, one or more of the PEG molecules in the branched structuresdescribed above can be replaced with a different polymer type.

The polymer can also be prepared with one or more hydrolytically stableor degradable linkages in the polymer backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. Other hydrolytically degradable linkages that may beincorporated include carbonate, imine, phosphate ester, hydrazone,acetal, orthoester, and phosphoramidate linkages.

The term poly(ethylene glycol) or PEG includes any or all the abovedescribed variations. Generally preferred PEG structures include linearmonofunctional, branched monofunctional, and linear, branched or forkeddifunctional or trifunctional PEGs.

Because end-capped polyethylene glycol starting materials, such as mPEG(methoxy-PEG) or bPEG (benzyloxy-PEG), can contain detectable amounts ofPEG diol impurity, leading to side products that are often difficult toanalyze or separate, the PEG starting material is, in one preferredembodiment, a diol-free benzyloxy-PEG as described in co-owned U.S. Pat.No. 6,448,369.

IV. Further Derivatization and Conjugation of Carboxylic AcidFunctionalized Polymers

A. Overview

If desired, a carboxylic acid functionalized polymer prepared by themethod of the invention can be further modified to form useful reactivederivatives of carboxylic acids using methodology known in the art.Preparation of such derivatives is facilitated by the high purity of thecarboxylic acid functionalized polymers of the invention, as compared toprior art products containing, for example, residual starting materialpolymer and/or residual reagents such as TFA. This is a significantbenefit, particularly for a pharmaceutical product, since the presenceand amounts of such contaminants can be highly variable, thus leading toirreproducibility of the product.

Accordingly, the method of the invention, wherein a carboxylic acidfunctionalized polymer is prepared, may further comprise the steps of(i) modifying the carboxylic acid to form a reactive derivative and (ii)conjugating the reactive derivative to a pharmacologically relevantmolecule having a corresponding reactive functional group. The steps (i)and (ii) may be performed in situ, where the carboxylic acid isconverted to an activated derivative using one of many activatingreagents known in the art, then immediately reacted with the molecule tobe conjugated.

The carboxylic acid can be derivatized to form, for example, acylhalides, acyl pseudohalides, such as acyl cyanide, acyl isocyanate, andacyl azide, neutral salts, such as alkali metal or alkaline-earth metalsalts (e.g. calcium, sodium, or barium salts), esters, anhydrides,amides, imides, hydrazides, and the like. In a preferred embodiment, theacid is esterified to form an active ester, such as an N-succinimidylester, o-, m-, or p-nitrophenyl ester, 1-benzotriazolyl ester,imidazolyl ester, or N-sulfosuccinimidyl ester.

In one embodiment, the further derivatized polymer is a PEG polymerhaving the structure:mPEG-O—(CR¹R²)_(n)—C(═O)—Z  (V)wherein R¹, R² and n are as described above. The moiety Z is preferablyselected from the group consisting of halo, amino, substituted amino,—NCO, —NCS, N₃, —CN, and —OR′, wherein R′ is selected fromN-succinimidyl, nitrophenyl, benzotriazolyl, imidazolyl,N-sulfosuccinimidyl, N-phthalimidyl, N-glutarimidyl,N-tetrahydrophthalimidyl, N-norbornene-2,3-dicarboximidyl, andhydroxy-7-azabenzotriazolyl.

The carboxyl-containing polymer produced by the method of the invention,or a reactive derivative thereof, can be used to form conjugates withbiologically active molecules, particularly biologically activemolecules carrying nucleophilic functional groups, such as amino,hydroxyl, or mercapto (thiol) groups.

Frequently, the molecule to be conjugated is a protein. Proteins areconjugated via reactive amino acids, such as lysine, histidine,arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine,cysteine, the N-terminal amino group, and the C-terminal carboxylicacid. Carbohydrate moieties on glycosylated proteins may also beemployed as conjugation sites. For reaction with an activated carboxylicacid, the most suitable groups are the N-terminal amino group,amine-containing side chains on lysine, histidine, and arginine,hydroxyl-containing side chains on serine, threonine, and tyrosine, andthiol side chains on cysteine.

Although the preferred methods of conjugation of the carboxyl-containingpolymers of the invention employ activated carboxylic acid derivatives,which react with nucleophilic groups on the molecule to be conjugated,it is also possible to derivatize the terminal carboxyl group to containany variety of functional groups. For example, in one embodiment, themoiety Z in structure (V) above has the structure —NHR₆, wherein R₆ isan organic group that contains a reactive functional group (e.g.,aldehyde, maleimide, mercapto, hydroxyl, amino, etc.), the functionalgroup(s) being separated from the nitrogen atom by an alkylene chain(e.g., C1-6) and, optionally, an additional linker, such as a short PEGchain and another alkylene chain (e.g., alkylene-PEG-alkylene).

B. Exemplary Methods of Conjugation

Such polymer conjugates can be formed using known techniques forcovalent attachment of an activated polymer, such as an activated PEG,to a biologically active agent. See, for example, Poly(ethylene glycol):Chemistry and Biological Applications, J. M. Harris and S. Zalipsky,editors, American Chemical Society, Washington, D.C. (1997) orBioconjugate Techniques, G. T. Hermanson, Academic Press (1996). Ingeneral, conjugation reactions are typically carried out in a buffer,such as a phosphate or acetate buffer, at or near room temperature,although conditions will depend on the particular reaction being carriedout. An excess of the polymeric reagent is typically combined with theactive agent. In some cases, however, it is preferred to havestoichiometric amounts of the reactive groups on the polymeric reagentand on the active agent.

Progress of a conjugation reaction can be monitored by SDS-PAGE,MALDI-TOF mass spectrometry, or any other suitable analytical method.Once a plateau is reached with respect to the amount of conjugate formedor the amount of unconjugated polymer remaining, the reaction is assumedto be complete. The product mixture is purified, if necessary, toseparate excess reagents, unconjugated reactants (e.g., active agent)undesired multi-conjugated species, and/or unreacted polymer, usingknown methods.

For example, conjugates having different molecular weights can beseparated using gel filtration chromatography. Fractions may be analyzedby a number of different methods, e.g. (i) OD at 280 nm for proteincontent, (ii) BSA protein analysis, (iii) iodine testing for PEG content(Sims et al., Anal. Biochem. 107:60-63, 1980), or (iv) SDS-PAGE,followed by staining with barium iodide.

Separation of positional isomers (that is, conjugates of the same orsubstantially the same molecular weight having a polymer attached atdifferent positions on a molecule) can be carried out by reverse phaseHPLC or ion exchange chromatography.

The conjugated product may be lyophilized for storage, with or withoutresidual buffer. In some instances, it is preferable to exchange abuffer used for conjugation, such as sodium acetate, for a volatilebuffer, such as ammonium carbonate or ammonium acetate, that can bereadily removed during lyophilization. Alternatively, a buffer exchangestep may be used using a formulation buffer, so that the lyophilizedconjugate is in a form suitable for reconstitution into a formulationbuffer and ultimately for administration to a mammal.

C. Exemplary Agents for Conjugation

A biologically active agent for use in coupling to polymer formed by themethod of the invention may be any one or more of the following.Suitable agents may be selected from, for example, hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxidants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules,peptides, polypeptides, proteins, antibodies, polysaccharides, steroids,nucleotides, oligonucleotides, polynucleotides, fats, electrolytes, andthe like. Preferably, an active agent for coupling to acarboxyl-containing polymer of the invention possesses a native amino,hydroxyl, or thiol group, or is modified to contain at least one suchgroup.

Specific examples of active agents include but are not limited toaspariginase, amdoxovir (DAPD), antide, becaplermin, calcitonins,cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists(e.g., peptides from about 10-40 amino acids in length and comprising aparticular core sequence as described in WO 96/40749), dornase alpha,erythropoiesis stimulating protein (NESP), coagulation factors such asFactor V, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X,Factor XII, Factor XIII, von Willebrand factor; ceredase, cerezyme,alpha-glucosidase, collagen, cyclosporin, alpha defensins, betadefensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones, human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interluekin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosponates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracehular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer includeamifostine, amiodarone, aminocaproic acid, aminohippurate sodium,aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amsacrine,anagrelide, anastrozole, asparaginase, anthracyclines, bexarotene,bicalutamide, bleomycin, buserelin, busulfan, cabergoline, capecitabine,carboplatin, carmustine, chlorambucin, cilastatin sodium, cisplatin,cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine,camptothecins, 13-cis retinoic acid, all trans retinoic acid,dacarbazine, dactinomycin, daunorubicin, deferoxamine, dexamethasone,diclofenac, diethylstilbestrol, docetaxel, doxorubicin, epirubicin,estramustine, etoposide, exemestane, fexofenadine, fludarabine,fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine,epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib,irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole,lisinopril, lovothyroxine sodium, lomustine, mechlorethamine,medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminolbitartrate, methotrexate, metoclopramide, mexiletine, mitomycin,mitotane, mitoxantrone, naloxone, nicotine, nilutamide, octreotide,oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, granicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. The above biologically activeproteins are additionally meant to encompass variants having one or moreamino acids substituted (e.g., cysteine), deleted, or the like, as longas the resulting variant protein possesses at least a certain degree ofactivity of the parent (native) protein.

V. Pharmaceutical Compositions and Administration Methods

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Exemplary excipients include, withoutlimitation, those selected from the group consisting of carbohydrates,antimicrobial agents, antioxidants, surfactants, buffers, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumarate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects. Generally, however, the excipient will bepresent in the composition in an amount of about 1% to about 99% byweight, preferably from about 5%-98% by weight, more preferably fromabout 15-95% by weight of the excipient, with concentrations less than30% by weight most preferred.

The foregoing pharmaceutical excipients and others are described inRemington: The Science & Practice of Pharmacy, 19′ ed., Williams &Williams, (1995), the Physician's Desk Reference, 52^(nd) ed., MedicalEconomics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook ofPharmaceutical Excipients, 3^(rd) Edition, American PharmaceuticalAssociation, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate, as determined by those skilledin the art. The method comprises administering, generally via injection,a therapeutically effective amount of the conjugate, preferably providedas part of a pharmaceutical preparation.

The actual dose to be administered will vary depend upon the age,weight, and general condition of the subject, as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and the conjugate being administered. Therapeuticallyeffective amounts of particular drugs are known to those skilled in theart and/or are described in the pertinent reference texts andliterature. Generally, a therapeutically effective amount of conjugatewill range from about 0.001 mg to 100 mg, preferably in doses from 0.01mg/day to 75 mg/day, and more preferably in doses from 0.10 mg/day to 50mg/day. The unit dosage of any given conjugate (again, preferablyprovided as part of a pharmaceutical preparation) can be administered ina variety of dosing schedules depending on the judgment of theclinician, needs of the patient, and so forth.

EXAMPLES

The following examples are provided to illustrate the invention butshould not be considered in limitation of the invention. For example,although PEG is used in the Examples, the use of other water soluble,non-peptidic polymers is encompassed by the invention, as discussedabove.

All PEG reagents referred to in these Examples are available from NektarAL, Huntsville, Ala. All NMR data was generated by a 300 or 400 MHz NMRspectrometer manufactured by Bruker.

Example 1 illustrates reaction of mPEG-OH with tert-butyl bromoacetatein the presence of a base to form a tert-butyl ester terminated polymer.Thereafter, the polymer is subjected to base-promoted hydrolysis usingNaOH as the base, followed by acidification using phosphoric acid, toform the final carboxylic acid terminated polymer.

Examples 2 and 3 exemplify similar reaction of a difunctional PEGstarting material (PEG diol; HO-PEG-OH). Example 4 illustrates reactionof a multifunctional, 4-armed PEG starting material, based on apentaerythritol core and having four reactive hydroxyls, one at theterminus of each PEG “arm”.

Example 1 mPEG(30,000)-carboxylic acid

A solution of mPEG-30,000 (50 g, 0.00167 moles) (NOF Corporation) intoluene (600 ml) was azeotropically dried by distilling off 300 mltoluene. t-Butanol (70 ml), potassium tert-butoxide (95%, 1.75 g, 0.0148moles, 8.9 fold excess) and tert-butyl bromoacetate (3.3 g, 0.0169moles, 10.1 fold excess) were added, and the mixture was stirredovernight at 45° C. under argon atmosphere. The solvent was distilledoff under reduced pressure, and the residue was dissolved in distilledwater (1000 ml).

The pH of the aqueous solution was adjusted to 12 with 1 M sodiumhydroxide, and the solution was stirred for 18 h, keeping the pH at 12by periodic addition of 1M sodium hydroxide.

The pH was adjusted to 3 with 5% phosphoric acid, and the product wasextracted with dichloromethane. The extract was dried with anhydrousmagnesium sulfate and added to ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure, giving a yield of 46.6 g.

NMR (d₆-DMSO): 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.01 ppm(s, —CH₂—COO—).

Anion exchange chromatographic analysis: mPEG(30,000)-carboxylic acid100%. This analysis showed that essentially no starting material orother polymeric impurity was present in the ether-precipitated product.

Example 2 PEG(10,000)-dicarboxylic acid

PEG-10,000 (35.25 g, 0.00705 eq) (NOF Corporation) (terminated at bothends with hydroxyl) was dissolved in toluene (600 ml) and azeotropicallydried by distilling off toluene. The residue was redissolved intoanhydrous toluene (500 ml). tert-Butanol (40 ml), potassiumtert-butoxide (4 g, 0.0356 moles, 5.1 fold excess) and anhydrous toluene(40 ml) were combined and added to the above reaction mixture, followedby stirring for about 3.5 hours. t-Butyl bromoacetate (7 ml, 0.0474moles, 6.7 fold excess) was added, and the mixture was stirred overnightat 40 degree C. under argon atmosphere. The solvent was distilled offunder reduced pressure, and the residue was dissolved in distilled water(1000 ml).

The pH of the aqueous solution was adjusted to 12.1 with 1M sodiumhydroxide, and the solution was stirred overnight, keeping the pH at12.1 by periodic addition of 1M sodium hydroxide.

The pH was adjusted to 1.0 with 1M hydrochloric acid, and the productwas extracted with dichloromethane. The extract was dried with anhydroussodium sulfate, concentrated, and added to ethyl ether. The precipitatedproduct was filtered off and dried under reduced pressure, to yield 33g.

NMR (d₆-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s, —CH₂—COO—).

Anion exchange chromatographic analysis: PEG(10,000)-dicarboxylic acid100%.

Example 3 PEG(5,000)-dicarboxylic acid

A solution of PEG-5,000 (35 g, 0.01400 equivalents) (NOF Corporation) inacetonitrile (800 ml) was azeotropically dried by distilling offacetonitrile, and the residue was redissolved into anhydrous toluene(300 ml). t-Butanol (50 ml), potassium tert-butoxide (4.7 g, 0.0419moles, 2.99 fold excess), and anhydrous toluene (50 ml) were combinedand added to the above reaction mixture, followed by about 3.5 hours ofstirring. t-Butyl bromoacetate (7.2 ml, 0.0488 moles, 3.48 fold excess)was added, and the mixture was stirred 20 hrs at room temperature underan argon atmosphere. The solvent was distilled off under reducedpressure, and the residue was dissolved in distilled water (1000 ml).

The pH of the aqueous solution was adjusted to 12.0 with 1M sodiumhydroxide, and the solution was stirred overnight, keeping the pH at12.0 by periodic addition of 1M sodium hydroxide.

The pH was adjusted to 2.0 with 1M hydrochloric acid and the product wasextracted with dichloromethane. The extract was dried with anhydroussodium sulfate, concentrated and added to ethyl ether. The precipitatedproduct was filtered off and dried under reduced pressure, to yield 32g.

NMR (d₆-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s, —CH₂—COO—).

Anion exchange chromatographic analysis: PEG(5,000)-dicarboxylic acid100%.

Example 4 4-Arm-PEG(10,000)-tetracarboxylic acid

A solution of Multi-arm PEG (4-Arm), MW 10 kDa (Nektar, Huntsville Ala.)(160 g, 0.064 equivalents) in toluene (2,300 ml) was azeotropicallydried by distilling off 1,000 ml of toluene at 80° C. under reducedpressure. In another vessel, tert-butanol (17.3 ml) and potassiumtert-butoxide (7.18 g, 0.128 moles, 2.00 fold excess) were mixed andthen added to the dried toluene solution from above. The resultingsolution was stirred for about 3.5 hours at 45° C. t-Butyl bromoacetate(20.8 ml, 0.141 moles, 2.20 fold excess) was added, and the mixture wasstirred 12 hrs at 45° C. under an argon atmosphere. The solvent wasdistilled off under reduced pressure, and the residue was dissolved indistilled water (1,600 ml).

The pH of the aqueous solution was adjusted to 12.0 with 1M sodiumhydroxide, and the solution was stirred for 17 hr while keeping the pHat 12.0 by periodic addition of 1M sodium hydroxide.

The pH was then adjusted to 1.5 with 1M phosphoric acid, and the productwas extracted with dichloromethane. The extract was dried with anhydroussodium sulfate, concentrated and added to ethyl ether. The precipitatedproduct was filtered off and dried under reduced pressure, to yield 15.5g.

NMR (d₆-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s, —CH₂—COO—),substitution 100%.

After 8 months of storage at −20° C., GPC analysis was identical to theoriginal product. Therefore, no detectable degradation occurred duringstorage.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, the invention is not to be limited to the specificembodiments disclosed, and modifications and other embodiments areintended to be included, within the scope of the appended claims.

It is claimed:
 1. A method of preparing a carboxylic-acid functionalizedmethoxypolyethylene glycol (m PEG), comprising: (i) reacting, in anorganic solvent, a two-fold up to a 30-fold molar excess of a tertiaryester reagent having a structure,

 where X is a halo group, and each of R³, R⁴ and R⁵ is phenyl, with amethoxyPEG-OH (mPEG-OH) having a molecular weight selected from 10,000Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da and 40,000 Da, in thepresence of base at a temperature of about 25-50° C., to thereby form areaction mixture comprising a mPEG-tertiary ester having a structure,

(ii) removing the organic solvent from the reaction mixture bydistillation to provide a residue comprising the mPEG-tertiary ester,(iii) hydrolyzing the mPEG-tertiary ester by addition of a strong basein aqueous solution to the residue from (ii) to thereby form amPEG-carboxylate salt, (iv) directly treating, without furtherisolation, the mPEG carboxylate salt from (iii) with aqueous inorganicacid to thereby provide a reaction mixture comprising a carboxylicacid-functionalized mPEG having a structure, mPEG-OCH₂C(O)OH, and (v)isolating the carboxylic acid-functionalized mPEG from the reactionmixture, wherein the isolated carboxylic acid-functionalized mPEGcontains less than 5% by weight of mPEG-OH, wherein no trifluoroaceticacid is used in the process and the methoxyPEG-OH is branched.
 2. Themethod of claim 1, wherein X is selected from bromo, chloro and iodo. 3.The method of claim 1, wherein the organic solvent is selected fromt-butanol, benzene, toluene, xylenes, tetrahydrofuran (THF),dimethylformamide (DMF), and dimethylsulfoxide (DMSO).
 4. The method ofclaim 1, wherein the base in step (i) is selected from the groupconsisting of potassium t-butoxide, butyl lithium, sodium amide, andsodium hydride.
 5. The method of claim 1, wherein the reacting step iscarried out for from about 0.5 hours to about 24 hours.
 6. The method ofclaim 1, wherein the hydrolyzing step is carried out at a pH of about 9or above.
 7. The method of claim 6, wherein the hydrolyzing step iscarried out at a pH of about 11 to about
 13. 8. The method of claim 6,wherein the strong base in the hydrolyzing step is an alkali metalhydroxide.
 9. The method of claim 8, wherein the alkali metal hydroxideis either sodium hydroxide or potassium hydroxide.
 10. The method ofclaim 1, wherein the hydrolyzing step is carried out at a temperature ofabout 10-30° C.
 11. The method of claim 1, wherein the treating step(iv) is effective to produce a reaction mixture with a pH of about 2 to3.
 12. The method of claim 11, wherein the aqueous inorganic acid fromstep (iv) is selected from sulfuric acid, nitric acid, phosphoric acidand hydrochloric acid.
 13. The method of claim 1, wherein treating step(iv) is carried out at a temperature from about 10° C. to about 30° C.14. The method of claim 1, wherein the isolated carboxylicacid-functionalized mPEG contains less than 2% by weight of mPEG-OH. 15.The method of claim 1, wherein the methoxyPEG-OH has a molecular weightof 20,000 Da or 40,000 Da.
 16. The method of claim 1, wherein theaqueous inorganic acid is phosphoric acid.